A therapy for dengue is still elusive. We describe the neutralizing and protective capacity of a dengue serotype-cross-reactive antibody isolated from the plasmablasts of a patient. Antibody SIgN-3C neutralized all four dengue virus serotypes at nano to picomolar concentrations and significantly decreased viremia of all serotypes in adult mice when given 2 days after infection. Moreover, mice were protected from pathology and death from a lethal dengue virus-2 infection. To avoid potential Fc-mediated uptake of immune complexes and ensuing enhanced infection, we introduced a LALA mutation in the Fc part. SIgN-3C-LALA was as efficient as the non-modified antibody in neutralizing dengue virus and in protecting mice while antibody-dependent enhancement was completely abrogated. The epitope of the antibody includes conserved amino acids in all three domains of the glycoprotein, which can explain its cross-reactivity. SIgN-3C-LALA neutralizes dengue virus both pre and post-attachment to host cells. These attributes likely contribute to the remarkable protective capacity of SIgN-3C.

The pathogenesis of severe dengue remains unclear, particularly the mechanisms underlying the plasma leakage that results in hypovolaemic shock in a small proportion of individuals. Maximal leakage occurs several days after peak viraemia implicating immunological pathways. Skin is a highly vascular organ and also an important site of immune reactions with a high density of dendritic cells (DCs), macrophages and T cells. We obtained skin biopsies and contemporaneous blood samples from patients within 24 hours of onset of dengue shock syndrome (DSS), and from healthy controls. We analyzed cell subsets by flow cytometry, and soluble mediators and antibodies by ELISA; the percentage of migratory CD1a(+) dermal DCs was significantly decreased in the DSS patients, and skin CD8(+) T cells were activated, but there was no accumulation of dengue-specific antibodies. Inflammatory monocytic cells were not observed infiltrating the skin of DSS cases on whole-mount histology, although CD14(dim) cells disappeared from blood.

Depending on the tissue microenvironment, T cells can differentiate into highly diverse subsets expressing unique trafficking receptors and cytokines. Studies of human lymphocytes have primarily focused on a limited number of parameters in blood, representing an incomplete view of the human immune system. Here, we have utilized mass cytometry to simultaneously analyze T cell trafficking and functional markers across eight different human tissues, including blood, lymphoid, and non-lymphoid tissues. These data have revealed that combinatorial expression of trafficking receptors and cytokines better defines tissue specificity. Notably, we identified numerous T helper cell subsets with overlapping cytokine expression, but only specific cytokine combinations are secreted regardless of tissue type. This indicates that T cell lineages defined in mouse models cannot be clearly distinguished in humans. Overall, our data uncover a plethora of tissue immune signatures and provide a systemic map of how T cell phenotypes are altered throughout the human body.

Recently it was found that the erythropoietin receptor (EpoR) is expressed on innate immune cells, such as dendritic cells and macrophages. We found that murine bone marrow-derived mast cells express the EpoR and that its expression is increased under hypoxic conditions. Interestingly, Epo stimulation of the cells did not activate signal transducer and activator of transcription molecules, nor did we find differences in the expression of typical STAT-dependent genes, the proliferation rate, and the ability to differentiate or to protect the cells from apoptosis. Instead, we demonstrate that stimulation of mast cells with Epo leads to phosphorylation of the receptor tyrosine kinase c-kit. We hypothesize that this is due to the formation of a receptor complex between the EpoR and c-kit. The common beta chain of the IL-3 receptor family, which was described as part of the tissue protective receptor (TPR) on other non-erythroid cells, however is not activated. To investigate whether the EpoR/c-kit complex has tissue protective properties, cells were treated with the Toll-like receptor ligand LPS. Combined Epo and LPS treatment downregulated the inflammatory response of the cells as detected by a decrease in IL-6 and TNF-α secretion.

RNA interference (RNAi) has become an established tool for basic gene annotation and a promising option for therapeutic concepts that require specific inhibition of dysregulated or exogenous genes. However, we and others accumulated strong evidence that overexpression of short hairpin RNAs (shRNAs, an expressable and widely used form of RNAi trigger) can cause cytotoxicity leading up to organ damage and lethality in shRNA-treated animals. A meticulous dissection of the underlying mechanisms is critical as it will ultimately guide the design of safer RNAi therapies and thus foster their clinical translation. One informative finding was that co-expression of Ago2 (Slicer, as it cleaves targeted mRNA), a key component of the RNA-induced silencing complex RISC, increases shRNA efficiency in cells and animals, implying that Ago2 is a rate-limiting factor whose saturation may be involved in cytotoxicity. Moreover, over-expression of the three other mammalian Ago proteins – Ago1, Ago3 or Ago4 (all slicing-incompetent) – dampens RNAi potency, underscoring that a delicate balance of all four Ago variants is key to efficient and safe gene silencing. Here, we investigated three hypotheses that could explain how relative Ago1-4 levels may affect the functionality of shRNA-loaded RISC and hence the strength of target knockdown: over-abundance of non-Slicer Ago proteins could (i) dysregulate Ago2 expression (transcription and/or translation), (ii) re-localize intracellular Ago2 away from sites of RNAi activity, or (iii) quantitatively sequester shRNAs into slicing-incompetent RISC. Using codon-optimized Ago variants that are distinguishable by real-time PCR from the endogenous counterparts, as well as Western blotting, we could eliminate model (i). Likewise, wide-field and confocal microscopy analyses revealed no evidence for Ago2 re-localization upon Ago1/3/4 over-expression and vice versa. We therefore focused on hypothesis (iii) and fused the Ago1-4 cDNAs with GFP or RFP tags that enabled their specific immunoprecipitation, alone or in combination, using the TRAP system. Indeed, we found that Slicer and non-Slicers compete for shRNA loading and mRNA knockdown in a dose-dependent manner, readily explaining all prior observations of RNAi enhancement by Ago2 over-expression, or shRNA inhibition by Ago1/3/4, respectively. Importantly, the combination of GFP/RFP-Ago fusion constructs with the co-precipitation protocols newly established during this work should be widely useful to address other vital questions in the RNAi field, such as the selective processing of different cellular small RNAs by the four Ago proteins. Moreover, our notion that over-expression of non-Slicers has no adverse effect on endogenous Ago2 levels or localization paves the way for novel strategies to fine-tune therapeutic RNAi in a safe and potent manner. As a prototype, we present a new shRNA/Ago co-expression system whereby Ago1/2 levels can be regulated independently through exogenous macrolides, permitting a tight control of target knockdown efficiencies and providing an original avenue to reduce toxicity in future RNAi gene therapy applications.

Bacterial components are recognized by the immune system through activation of the inflammasome, eventually causing processing of the proinflammatory cytokine interleukin-1? (IL-1?), a pleiotropic cytokine and one of the most important mediators of inflammation, through the protease caspase-1. Synthesis of the precursor protein and processing into its bioactive form are tightly regulated, given that disturbed control of IL-1? release can cause severe autoinflammatory diseases or contribute to cancer development. We show that the bacterial Pasteurella multocida toxin (PMT) triggers Il1b gene transcription in macrophages independently of Toll-like receptor signaling through RhoA/Rho-kinase-mediated NF-?? activation. Furthermore, PMT mediates signal transducer and activator of transcription (STAT) protein-controlled granzyme A (a serine protease) expression in macrophages. The exocytosed granzyme A enters target cells and mediates IL-1? maturation independently of caspase-1 and without inducing cytotoxicity. These findings show that macrophages can induce an IL-1?-initiated immune response independently of inflammasome activity.

Regulatory B cells (B-reg) produce IL-10 and suppress inflammation in both mice and humans, but limited data on the phenotype and function of these cells have precluded detailed assessment of their contribution to host immunity. In this article, we report that human B-reg cannot be defined based on a phenotype composed of conventional B cell markers, and that IL-10 production can be elicited in both the CD27+ memory population and naive B cell subset after only a brief stimulation in vitro. We therefore sought to obtain a better definition of IL-10–producing human B-regs using a multiparameter analysis of B cell phenotype, function, and gene expression profile. Exposure to CpG and anti-Ig are the most potent stimuli for IL-10 secretion in human B cells, but microarray analysis revealed that human B cells cotreated with these reagents resulted in only ∼0.7% of genes being differentially expressed between IL-10+ and IL-10− cells. Instead, connectivity map analysis revealed that IL-10–secreting B cells are those undergoing specific differentiation toward a germinal center fate, and we identified a CD11c+ B cell subset that was not capable of producing IL-10 even under optimal conditions. Our findings will assist in the identification of a broader range of human pro–B-reg populations that may represent novel targets for immunotherapy.

ABSTRACT Dengue virus (DENV) infects an estimated 400 million people every year, causing prolonged morbidity and sometimes mortality. Development of an effective vaccine has been hampered by the lack of appropriate small animal models; mice are naturally not susceptible to DENV and only become infected if highly immunocompromised. Mouse models lacking both type I and type II interferon (IFN) receptors (AG129 mice) or the type I IFN receptor (IFNAR−/− mice) are susceptible to infection with mouse-adapted DENV strains but are severely impaired in mounting functional immune responses to the virus and thus are of limited use for study. Here we used conditional deletion of the type I IFN receptor (IFNAR) on individual immune cell subtypes to generate a minimally manipulated mouse model that is susceptible to DENV while retaining global immune competence. Mice lacking IFNAR expression on CD11c+ dendritic cells and LysM+ macrophages succumbed completely to DENV infection, while mice deficient in the receptor on either CD11c+ or LysM+ cells were susceptible to infection but often resolved viremia and recovered fully from infection. Conditional IFNAR mice responded with a swift and strong CD8+ T-cell response to viral infection, compared to a weak response in IFNAR−/− mice. Furthermore, mice lacking IFNAR on either CD11c+ or LysM+ cells were also sufficiently immunocompetent to raise a protective immune response to a candidate subunit vaccine against DENV-2. These data demonstrate that mice with conditional deficiencies in expression of the IFNAR represent improved models for the study of DENV immunology and screening of vaccine candidates. IMPORTANCE Dengue virus infects 400 million people every year worldwide, causing 100 million clinically apparent infections, which can be fatal if untreated. Despite many years of research, there are no effective vaccine and no antiviral treatment available for dengue. Development of vaccines has been hampered in particular by the lack of a suitable small animal model. Mouse models used to test dengue vaccine are deficient in interferon (IFN) type I signaling and severely immunocompromised and therefore likely not ideal for the testing of vaccines. In this study, we explored alternative models lacking the IFN receptor only on certain cell types. We show that mice lacking the IFN receptor on either CD11c- or LysM-expressing cells (conditional IFNAR mice) are susceptible to dengue virus infection. Importantly, we demonstrate that conditional IFN receptor knockout mice generate a better immune response to live virus and a candidate dengue vaccine compared to IFNAR mice and are resistant to subsequent challenge.

Dengue is a growing global concern with 390 million people infected each year. Dengue virus (DENV) is transmitted by mosquitoes, thus host cells in the skin are the first point of contact with the virus. Human skin contains several populations of antigen-presenting cells which could drive the immune response to DENV in vivo: epidermal Langerhans cells (LCs), three populations of dermal dendritic cells (DCs), and macrophages. Using samples of normal human skin we detected productive infection of CD14(+) and CD1c(+) DCs, LCs and dermal macrophages, which was independent of DC-SIGN expression. LCs produced the highest viral titers and were less sensitive to IFN-β. Nanostring gene expression data showed significant up-regulation of IFN-β, STAT-1 and CCL5 upon viral exposure in susceptible DC populations. In mice infected intra-dermally with DENV we detected parallel populations of infected DCs originating from the dermis and migrating to the skin-draining lymph nodes. Therefore dermal DCs may simultaneously facilitate systemic spread of DENV and initiate the adaptive anti-viral immune response.

ABSTRACT In a recent clinical trial, balapiravir, a prodrug of a cytidine analog (R1479), failed to achieve efficacy (reducing viremia after treatment) in dengue patients, although the plasma trough concentration of R1479 remained above the 50% effective concentration (EC50). Here, we report experimental evidence to explain the discrepancy between the in vitro and in vivo results and its implication for drug development. R1479 lost its potency by 125-fold when balapiravir was used to treat primary human peripheral blood mononuclear cells (PBMCs; one of the major cells targeted for viral replication) that were preinfected with dengue virus. The elevated EC50 was greater than the plasma trough concentration of R1479 observed in dengue patients treated with balapiravir and could possibly explain the efficacy failure. Mechanistically, dengue virus infection triggered PBMCs to generate cytokines, which decreased their efficiency of conversion of R1479 to its triphosphate form (the active antiviral ingredient), resulting in decreased antiviral potency. In contrast to the cytidine-based compound R1479, the potency of an adenosine-based inhibitor of dengue virus (NITD008) was much less affected. Taken together, our results demonstrate that viral infection in patients before treatment could significantly affect the conversion of the prodrug to its active form; such an effect should be calculated when estimating the dose efficacious for humans.